Functional conservation of the capacity for ent-kaurene biosynthesis and an associated operon in certain rhizobia

Abstract

Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants.

Fig 1

Production of ent-kaurene by B. japonicum. Shown are the steps catalyzed by the characterized ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (ent-KS).

FIG 2

Selected ion (m/z 272) chromatograms obtained by GC-MS demonstrating production of ent-kaurene from GGPP by coexpressing CPS and KS from M. loti (A), S. fredii (B), and R. etli (C), along with an authentic standard (from coexpression of the CPS and KS from Arabidopsis thaliana) in E. coli (along with a GGPP synthase) (D).

FIG 3

Schematic of diterpenoid biosynthesis operon from the designated rhizobia (with the gene designations described in the text).

FIG 4

Selected ion (m/z 272) chromatograms obtained by GC-MS demonstrating production of ent-kaurene from S. meliloti 1021 expressing GGPS-CPS-KS, but not CPS-KS alone, from S. fredii NGR234 (as indicated).

FIG 5

Molecular phylogenetic analysis of genes for the characterized rhizobial CPS (A) and genes for the nitrogenase subunit NifK (B) from the same rhizobia. SsCPS and AvNifK are the designated outgroup sequences, as described in the text. SfCPS, S. fredii CPS; MlCPS, M. loti CPS; ReCPS, R. etli CPS; ReNifK, R. etli NifK; MlNifK, M. loti NifK; SfNifK, S. fredii NifK; BjNifK, B. japonicum NifK.